This invention relates to a method and a device for the preparation of samples for ionization with matrix-assisted laser desorption. MALDI time-of-flight mass spectrometry is often used in clinical microbiology for the identification of microorganisms, particularly those that cause human infectious diseases. The principle of matrix-assisted laser desorption and ionization (MALDI) has already been described in detail elsewhere (see for example M. Karas et al. “Principles and applications of matrix-assisted UV-laser desorption/ionization mass spectrometry”, 1990, Analytica Chimica Acta, 241, 175-185). To explain it very briefly: in a widely used version of MALDI sample preparation, soluble analyte molecules are inserted into a structure of matrix crystals which have a high absorptivity for laser light. If the matrix crystal structure is exposed to pulses of laser radiation, it vaporizes explosively and releases the embedded analyte molecules. Analyte molecules are also ionized during this very energetic ablation process. These molecules are therefore available for a subsequent mass spectrometric analysis. For this analysis, the analyte ions are usually accelerated in electric fields to different velocities depending on their masses m and number z of charges, and after they have passed through a long flight path, which resolves them by their velocities, they are fed to a detector with a secondary electron multiplier. The timespans from the laser desorption of the sample material, or from an acceleration pulse in an orthogonal time-of-flight mass spectrometer, until the different ion current signals arrive at the detector give, via a time-to-mass transformation, the mass-to-charge ratios m/z of the analyte ions.
In clinical microbiology, microorganisms of clinical samples, such as mucosal smears, are cultured specifically in order to provide the minimum number of microorganisms which are required for the detection limit of the particular detection method, and to be able to repeat sample-consuming investigations, where necessary. To this end, the clinical sample is often applied to a nutrient medium such as agar. Under suitable culturing conditions, for example at controlled temperature and humidity, distinguishable colonies of the microorganisms are grown on the culture medium.
The usual method of MALDI sample preparation is to manually take up biological material of a single colony on a nutrient medium with an inoculation swab and transfer it to a sample site of a sample support as the analyte material. The biological material thus applied is subsequently wetted with a matrix solution. The matrix solution contains dissolved molecules of the matrix substance, which, as the drying process proceeds, form the crystals into which the analyte molecules are embedded. The matrix solution also contains a solvent which destroys the biological structure of the microorganism, particularly the cell wall. The decomposition process causes soluble material of the microorganism such as proteins or peptides to be released and brought into solution; these are the analyte molecules of actual interest and they are detected in the mass spectrometric analysis. Apart from these analyte molecules, cell residues also remain after decomposition. They are insoluble and thus impede the crystallization of the matrix material.
Owing to these omnipresent cell residues, the quantity of biological material which is transferred with the inoculation swab onto the sample site must be very small and within very narrow quantity tolerances for a mass spectrometric analysis. An optimum quantity is between around 10,000 and 100,000 cells, which usually occupy around one thousandth of a cubic millimeter. Such a quantity is very difficult to see with the naked eye. The insoluble cell residues which remain after cell decomposition as a type of fibrous pulp can impede the formation of a homogeneous crystal structure of the matrix material, especially one which is free from defects. But the matrix crystal structure must have a certain quality, especially in order to ensure that it has the desired absorption characteristics for laser light of a certain wavelength. Otherwise the ionization yield from the laser desorption is too low for informative time-of-flight mass spectra. In practice, the only solution up to now has been to ensure that a suitable quantity is smeared onto the sample site when depositing the biological material by hand. This demands a great deal of skill of the technician preparing the sample, who has to judge the quantity of biological material by eye in order to ensure that, on the one hand, the prepared sample is sufficiently abundant in terms of analyte molecules yet, on the other hand, is not impeded by too large an amount of unsuitable cell residues after the decomposition. It usually takes a long time for a technician to learn to assess how much biological material is suitable for MALDI sample preparation.
The problem of the remaining insoluble cell components which impede the formation of matrix crystals could be avoided if the decomposed cell material is filtered or centrifuged. Particularly when microorganisms are cultured in a liquid nutrient medium such as a blood or broth culture, filtration or centrifugation is performed several times in order to separate out insoluble cell residues, among other things. In contrast to a flat culture medium with colonies which can be spatially restricted, the growing microorganisms in the liquid nutrient medium are spatially dispersed. As a rule, after the growth phase of the microorganisms, the original components of the liquid nutrient medium, for example red blood cells, are selectively decomposed and then separated from the still largely intact cells of the microorganism with the aid of a first filtration or centrifugation. The microorganisms are subsequently destroyed in an extraction agent or solvent. With a further filtration or centrifugation, the insoluble cell components or cell residues of the microorganisms are then removed so that only the soluble cell components which were released by the destruction remain and are available for the sample preparation. This method of sample preparation is very time-consuming, however.
A further problem arises if the biological material to be analyzed requires a long decomposition time. Yeasts are an example of this. In this case, the extraction agent must act on the microorganism over a long period in order to not only destroy the substructure of the outer, resistant cell membranes but also to release the substructures of the cell interior which are actually of interest. If biological material is deposited onto a sample site of a sample support and then wetted with a matrix solution which is intended to effect the decomposition of the cells contained in the biological material, the time available for the matrix solution to act on the cells is limited by the vaporization of the solvent. When the solvent of the matrix solution has evaporated, the decomposition process ends, possibly before the interior of the cell could be extracted. In principle, the matrix solution with which the biological material is wetted could be dosed in a way which ensures the cells are completely decomposed before drying. However, the amounts of matrix solution required for this are found to be too great to be deposited on sample sites which usually cover an area of a few square millimeters. Increasing the area of the sample site whilst maintaining the current preparation practice would mean that a very small, possibly hardly perceptible, quantity of biological material would have to be smeared onto a disproportionately large area in comparison, which makes the positioning of the biological material more difficult, particularly if deposited manually.
In principle, where the biological material is highly resistant against decomposition, this can be countered with correspondingly more concentrated solvents, e.g. strong acids. However, strong solvents usually also impede the formation of a homogeneous matrix crystal structure, which is required for a high-yield desorption process. In order to avoid this problem, the decomposition can be carried out in two steps. In a first step, a strong extraction agent is added to the sample of biological material on the sample site; this completely decomposes the cell structure and is subsequently removed by evaporation. The soluble analyte molecules can then be dissolved again out of the remaining crust of insoluble and soluble cell components on the sample site by adding the conventional matrix solution, which does not decompose so strongly, and are embedded into the matrix which is forming on the sample site. Such a two-step preparation is a lengthy procedure, however, and again has the disadvantage that the insoluble cell components hinder the formation of a homogeneous layer of matrix crystals on the sample site, where both the decomposition and the matrix crystallization take place.
In view of the above, there is a need for a simplified sample preparation on a sample support for ionization with matrix-assisted laser desorption with which the problems with the matrix crystallization described above can be avoided.